Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membrane and secretory enzymes and other functional proteins seems essential. To that end, unique parasite surface membrane, secreted enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during the course of parasite growth, differentiation and development. For example, recently we identified and characterized the genes that encode the unique Leishmania secretory chitinase family. Using combined biochemical and molecular approaches we showed that this family of enzymes was functionally conserved among all pathogenic species of Leishmania examined. This suggested that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In addition, in previous studies, we demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. In that regard, in ongoing studies,we recently identified and characterized the biochemical and functional properties of a unique new, 35 kDa, nuclease from these organisms. Our studies demonstrated that this enzyme was constitutively released/secreted by both developmental forms of the parasite. Using a molecular approach, we identified, characterized and episomally-expressed the gene, LdNuc[S] which encodes this new Class I nuclease family member from these organisms. Results of both structural and functional analyses showed that the LdNuc[S] expressed-enzyme had properties identical to the native, wild-type, parasite released/secreted nuclease. Further, both LdNuc[S] mRNA and secretory nuclease activity were differentially up-expressed by amastigotes >> promastigotes. Biochemical analyses showed that the LdNuc[S] secreted nuclease could hydrolyze a variety of synthetic polynucleotide substrates as well as, both single- and double-stranded DNAs and RNAs. Based on these observations, we hypothesize that this leishmanial ?secretory? nuclease could act, at a distance away from the parasite, to hydrolyze host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, LdNuc[S] must have essential roles in facilitating the survival, growth and development of this important human pathogen. In other biochemical and molecular studies, we demonstrated that a unique L. donovani Rab5b-protein functions in identifying and maintaining the structural identity and integrity of early endosomal compartments in this parasite. We found that this protein helps to facilitate the proper endosomal trafficking and transit of both secretory and surface membrane proteins into and through the unique endocytic compartments of these parasites. Further, using site-specific GFP tagged-constructs, we showed that the Ld Rab5b was essential for the normal endocytic trafficing in these organisms. In addition, in collaborative studies we succeeded in identifying and characterizing a gene encoding a specific blood-induced chitinolytic enzyme system in the midgut of Phlebotomus papatasi and Lutzomyia longipalpis which are the sandfly vectors for Leishmania major and L. (donovani) chagasi, respectively. A recombinant expressed protein from this gene demonstrated high chitinolytic enzyme activity against a variety of chitin substrates. This is the first chitinase gene to be described from these important vectors for human parasites. Cumulatively, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.